Several fibroblast cell lines from patients with the rare lysosomal storage disease mucolipidosis II (I-cell disease) have been found to possess markedly elevated intracellular levels of cystine. In contrast, the intracellular cystine contents of two cell lines from patients with the similar, but clinically less severe, mucolipidosis III were within normal limits. The identity of excess free cystine in I-cells was confirmed by several independent assays including automated amino acid analysis, high-voltage paper electrophoresis, and by a specific cystine-binding protein assay. The finding of elevated cystine in I-cells may contribute to an understanding of the pathogenesis of human cystinosis. Studies on additional cell lines from patients with mucolipidosis II will be needed to determine whether elevated intracellular free cystine is an invariable concomitant of this disease.